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Abstract：
Joubert syndrome (JS) is a recessively inherited congenital ataxia characterized by hypotonia, psychomotor delay, abnormal ocular movements, intellectual disability, and a peculiar cerebellar and brainstem malformation, the \"molar tooth sign.\" Over 40 causative genes have been reported, all encoding for proteins implicated in the structure or functioning of the primary cilium, a subcellular organelle widely present in embryonic and adult tissues. In this paper, we developed an in vitro neuronal differentiation model using patient-derived induced pluripotent stem cells (iPSCs), to evaluate possible neurodevelopmental defects in JS. To this end, iPSCs from four JS patients harboring mutations in distinct JS genes (AHI1, CPLANE1, TMEM67, and CC2D2A) were differentiated alongside healthy control cells to obtain mid-hindbrain precursors and cerebellar granule cells. Differentiation was monitored over 31 days through the detection of lineage-specific marker expression by qRT-PCR, immunofluorescence, and transcriptomics analysis. All JS patient-derived iPSCs, regardless of the mutant gene, showed a similar impairment to differentiate into mid-hindbrain and cerebellar granule cells when compared to healthy controls. In addition, analysis of primary cilium count and morphology showed notable ciliary defects in all differentiating JS patient-derived iPSCs compared to controls. These results confirm that patient-derived iPSCs are an accessible and relevant in vitro model to analyze cellular phenotypes connected to the presence of JS gene mutations in a neuronal context.
摘要：
Joubert综合征（JS）是一种以张力过低为特征的隐性遗传性先天性共济失调，精神运动延迟，异常眼球运动，智力残疾,还有一种特殊的小脑和脑干畸形,“磨牙”标志。“已经报道了超过40个致病基因，所有编码与初级纤毛的结构或功能有关的蛋白质，一种广泛存在于胚胎和成体组织中的亚细胞器。在本文中,我们使用患者来源的诱导多能干细胞（iPSCs）开发了体外神经元分化模型，评估JS中可能的神经发育缺陷。为此,来自四名JS患者的iPSC在不同的JS基因（AHI1，CPLANE1，TMEM67和CC2D2A）中具有突变，与健康对照细胞一起分化，以获得中后脑前体和小脑颗粒细胞。通过qRT-PCR检测谱系特异性标志物表达，在31天内监测分化，免疫荧光，和转录组学分析。所有JS患者来源的iPSCs，不管突变基因是什么，与健康对照组相比，分化为中脑和小脑颗粒细胞的损伤相似。此外,初级纤毛计数和形态分析显示,与对照组相比,所有分化的JS患者来源的iPSCs均存在显著纤毛缺陷.这些结果证实，患者来源的iPSC是一种可利用的和相关的体外模型，用于分析与神经元环境中JS基因突变的存在相关的细胞表型。
